The PTEN tumor suppressor gene is a frequent target of somatic mutation, particularly in glioblastoma multiform and prostate cancer. The expression of PTEN in PTEN-mutant glioblastoma cells leads to a cell cycle arrest in G(0)/G(1) that is mediated at least partially by increased p27(kip1) levels. Here we show that p27(kip1) is not regulated by transcriptional control but that p27(kip1) protein shows increased stability after inhibition of the phosphoinositide (PI) 3-kinase pathway. Because p27(kip1) protein stability is known to be regulated by phosphorylation, we have examined modifications in the phosphorylation pattern after PI 3-kinase inhibition. Biochemical evidence suggests that p27(kip1) is phosphorylated on several serine residues, including Ser-10 and Ser-178, but that phosphorylation is unaltered by PI 3-kinase activity. This is further confirmed by the inducible expression of p27(kip1) phosphorylation site mutants, suggesting that p27(kip1) is destabilized in a phosphorylation-independent manner by the PI 3-kinase pathway at the G(1)/S transition.